Targeted therapeutics have gained prominence offering improved potency and reduced toxicity. There are two possibilities to specifically target an organ: first via a receptor expressed specifically in the target-organ or second via an enzyme with higher expression or higher activity in this organ. These concepts can be combined for even better targeting.
Tissue targeting is a critical topic of pharmaceutical investigation, in order to maximize the desired therapeutic effect whilst minimizing toxic side events, i.e. to improve the therapeutic index. Renal-specific drug targeting can be an attractive option to:                Avoid undesirable extra-renal effects        The intra-renal transport of a drug may not be optimal in relation to the target cell within the organ        Some drugs are largely inactivated before they reach the site of action in the kidneys        Pathological conditions such as abnormalities in glomerular filtration, tubular secretion, or the occurrence of proteinuria can affect the normal renal distribution of the drug.        
Furthermore, cell-specific drug targeting within the kidney may provide a tool in understanding certain mechanisms of drug action and thus also to manipulate renal physiology.
Within the kidney, injury to the podocytes is the initiating cause of many renal diseases, leading to proteinuria with possible progression to end-stage renal disease. The podocyte plays a key role both in maintenance of the glomerular filtration barrier and in glomerular structural integrity. Drug targeting to the podocyte imposes two important challenges: (1) to target the right cell (where an understanding of the anatomy of the podocyte and how it interacts with surrounding cells and structures is crucial) and (2) to select and address the right pharmacological pathway and which events lead to podocyte damage.
In parallel to podocyte injury leading to glomerulosclerosis, tubulointerstitial fibrosis is another hallmark of Acute Kidney Injury (AKI) and once again, drug targeting to the proximal tubular cells may offer new tools for its treatment, by reducing toxicity of drugs that exert unwanted side effects and/or by increasing the renal efficacy of antifibrotic drugs.
Recent advanced knowledge on renal diseases has yielded several candidate pathways for designing cell-targeted therapeutics, which include the Notch pathway. Within the kidney, injury to glomerular or tubular cells is the initiating cause of many acute and chronic diseases, leading to progressive dysfunction and end-stage renal disease. The glomerulus is the main filtration barrier that determines global kidney function. Inflammatory and non-inflammatory stresses affect the glomerulus and lead to alterations in its structure, thus in its permeability and function, leading to Chronic Kidney Disease (CKD). Injury to the tubulo-interstitial tissue is a major cause of Acute Kidney Injury (AKI) in particular in weakened hospitalized patients.
Acute Kidney Injury (AKI) is a risk factor for progressive to Chronic Kidney Disease (CKD), a serious clinical condition with no effective treatment, associated with patient's high morbidity including cardiovascular and metabolic complications. Kidney diseases can be primary or occur as part of multisystemic genetic, inflammatory, autoimmune, toxic or metabolic disorders. Three main structures can be affected in the kidney, the glomerulus, the tubulo-interstitial tissue or the vessels, leading to distinct clinical syndromes in the early stages of the disease. However, irrespective to the initial insult, disease progression will lead to irreversible glomerulosclerosis and tubulo-interstitial fibrosis, thus eventual end-stage renal disease requiring dialysis or kidney transplantation. Current therapies mainly aim at treating the primary disease to limit CKD progression. Previous studies have found that genes belonging to the Notch pathway were up-regulated in kidney samples from patients and in animal models of renal disease.
Notch is a membrane inserted protein, with its active part toward the intracellular space. The enzyme γ-secretase is a large protease complex composed of two aspartyl protease catalytic subunits (presenilin-1 and -2) and three support subunits (Pen-2, Aph-1 and nicastrin), all being membrane proteins. Substrates of γ-secretase first bind to nicastrin, then are transferred between the two presenilin subunits to the γ-secretase which performs an intra-membrane hydrolysis. The γ-secretase complex is able to activate Notch by hydrolyzing a peptide bond of the Notch protein at an intra-membrane site, allowing the cleaved Notch intracellular domain to migrate to the nucleus where it activates responsive genes. The intra-membrane activity of the γ-secretase has also been involved in the release from the membrane of other biologically relevant membrane proteins involved in normal and pathological processes, including insulin-like growth factor or sorting receptors. Therefore, for selective therapy, it is mandatory to achieve only localized inhibition of this activity, thus protecting the other functions of this enzyme and of the Notch pathway in particular.
The Notch pathway has been reported to participate in renal diseases and tissue damage in the kidney, thus being a potential target to treat renal diseases. However, as the γ-secretase and the Notch pathway are also important in controlling the function of other cells, including normal cells, it is necessary to develop tools for specifically targeting Notch antagonists to diseased kidney cells.
Genetic studies performed in mice with conditional expression of the active Notch1 protein showed massive glomerulosclerosis, leading ultimately to renal failure and death of the animals. Genetic deletion of Notch transcriptional binding partner as well as treatment with γ-secretase inhibitors, thus inhibiting the γ-secretase-mediated Notch activation and translocation to the nucleus, protected the animals from nephrotic syndrome. Thus, targeted pharmacologic inhibition of the Notch pathway signaling may prevent kidney damage in a variety of diseases. Regulation of the Notch pathway signaling mainly occurs at the levels of ligand binding and γ-secretase complex-mediated cleavage. Then, cleaved Notch migrates to the nucleus where it activates responsive genes, which include Notch1 itself.
γ-Secretase inhibiting compounds and preparation thereof have for example been described in WO 2006/061136 A2. Therein disclosed (6,7,8,9-tetrahydro-5-oxa-9-aza-benzocyclohepten-7-yl)-malonamide derivatives have been reported to be useful in the treatment of Alzheimer's disease or common cancers including but not limited to cervical carcinomas and breast carcinomas and malignancies of the hematopoietic system.
For the preparation of drug-carrier conjugates the commonly applied approach is to couple the drug covalently to the carrier. The characteristics of the bond between the drug and carrier system greatly influence the in vivo behavior of the drug-carrier conjugate. The conjugate needs to be stable in the circulation to prevent premature loss of the free drug, i.e. before the carrier has been accumulated in the intended target cells. But after its accumulation, quantitative release of the drug from the carrier and regeneration of the parent drug is desired.
Herein we describe a novel therapeutic strategy aimed to locally increase the concentration of γ-secretase inhibitors as Notch antagonists in the kidney by preparing prodrugs of γ-secretase inhibitor(s) coupled to substrates for specific hydrolytic enzyme-activities expressed at high levels by specific cells in injured kidneys. The ideal prodrug would be i) inactive on γ-secretase and ii) liberated only in the kidney to have no or very low exposure of parent in plasma, and e.g. in liver, to improve the safety (margin) window.
As is evidenced herein by in vitro assays as well as a mouse model of acute kidney tubulo-interstitial disease, the γ-secretase inhibiting pay-load is selectively liberated from the prodrug in the kidney exerting a regulatory effect on the Notch pathway. Our approach involves Ac-γ-Glu prodrugs of a γ-secretase inhibitor, targeting the γ-glutamyltranspeptidase (γ-GT) and/or a specific transporter in the kidney, and the intracellular N-acylamine acid deacylase (ACY1) and γ-glutamylcyclotransferase (γ-GCT) to selectively control the activation of the Notch pathway in the context of kidney disease. We could show that such an approach was able to improve the stability of the therapeutics and the selectivity for the diseased kidney of the designed prodrugs potentially opening the way for improving treatment and decreasing side-effects of therapies aimed at controlling the Notch pathway for patients with acute and chronic kidney disorders.